Introduction

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-adrenergic receptor (AR) stimulation plays a prominent role in modulation of cardiac myocyte performance in response to an increased peripheral demand. Driven by sympathetic neurotransmitters and adrenal hormones, AR activation regulates virtually all major steps of the cardiac cell excitation-contraction (E-C) coupling cascade, e.g., the sarcolemmal L-type Ca²⁺ current (I_Ca), sarcoplasmic reticulum (SR) Ca²⁺ release and reuptake, and the responsiveness of contractile myofilaments to cytosolic Ca²⁺. Because I_Ca provides the trigger for SR Ca²⁺ release, and is a major determinant of intracellular calcium homeostasis, modulation of this current by AR system has been extensively studied over the last two decades. It has been demonstrated that both ₁AR and ₂AR subtypes coexist in cardiac myocytes in many mammalian species, and that stimulation of each of these receptor subtypes increases cardiac I_Ca (Xiao and Lakatta, 1993; Cerbai et al., 1995) through the classic stimulatory G protein (G_s)-adenylyl cyclase-cAMP-protein kinase A (PKA) signaling cascade (Hartzell et al., 1991; Zhou et al., 1997; Skeberdis et al., 1997; Xiao et al., 1999). The existence and functional importance of a more rapid, direct interaction of the AR-activated G_s and L-type Ca²⁺ channel remain controversial (Yatani and Brown, 1989; Hartzell et al., 1991; Zhou et al., 1997; Skeberdis et al., 1997).

A prevailing receptor theory (two-state model) states that a G protein-coupled receptor, such as ₁AR or ₂AR, exists in an equilibrium between two conformational states: an inactive (R) state and an active (R*) state, the latter having high affinity for G proteins (Bond et al., 1995). In the absence of a receptor agonist, spontaneous transition between the R* and R states results in a constitutive or intrinsic activation of only minority of receptors (Chidiac et al., 1994; Bond et al., 1995) and thus the functional significance of R* is not always evident. The presence of a large number of spontaneously activated ₂ARs (₂-R*s), which alter basal function, has been experimentally demonstrated in a transgenic (TG) murine model, the TG4 mouse (Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999), in which the human ₂AR is overexpressed by ~200-fold in a cardiac-specific manner. Hence, this transgenic model provides a unique opportunity to study the transmembrane signal transduction originating from unliganded ₂-R* in comparison with that from the ligand-activated ₂AR (₂-LR*). According to the two-state receptor model, ₂-R* ought to be identical with ₂-LR*, because there is only a single active conformational state. However, there is no a priori reason that this has to be the case. By analogy to ionic channels and enzymes, it is more plausible that a receptor may possess multiple, distinct active conformations (Perez et al., 1996; Gurdal et al., 1997). If ₂-R* and ₂-LR* differ in their active conformational states, spontaneous and agonist-induced ₂-adrenergic signaling may not be functionally equivalent, e.g., in modulating their target proteins, such as L-type Ca²⁺ channels.

In the present study, we examined the possible modulatory effects of ₂-R* on basal I_Ca and cell contraction in single ventricular myocytes and on basal cAMP in myocardium from TG4 mice and wild-type (WT) littermates. Surprisingly, we found no evidence that I_Ca was regulated by ₂-R* in TG4 heart cells. In contrast, both ₂-LR* signaling in the presence of pertussis toxin (PTX) and direct adenylyl cyclase activation by forskolin augmented I_Ca to an extent similar to that observed in WT cells. Our results support the idea that despite many similarities, ₂-R* and ₂-LR* may represent distinct functional conformation states of the receptor, eliciting different intracellular signaling patterns, and having differential effects on target proteins. These findings require an extension of the current model of ₂AR to encompass multiple active conformational states.

	Experimental Procedures

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Cell Isolation and Measurement of Contraction. Single murine cardiac myocytes were isolated from the hearts of 2- to 3- month-old mice via a standard enzymatic technique (Korzick et al., 1997). Briefly, hearts were retrogradely perfused with collagenase B and protease using the Langendorff method. Cells were shaken loose from the heart after this perfusion and then suspended in HEPES buffer solution consisting of: 1 mM CaCl₂, 137 mM NaCl, 5.4 mM KCl, 15 mM dextrose, 1.3 mM MgSO₄, 1.2 mM NaH₂PO₄, and 20 mM HEPES, pH 7.4, adjusted with NaOH. Ca²⁺ tolerant cells were kept at 37°C, with or without incubation with 1.5 µg/ml PTX for at least 3 h, as described previously (Xiao et al., 1995).

Ca²⁺ Current Measurement. I_Ca was measured via the whole-cell patch clamp technique using an Axopatch 1D amplifier (Axon Instruments Inc., Foster City, CA). Low-resistance (1-2 M) micropipettes were pulled via a two-stage micropipette puller (model P-97; Sutter Instrument Co., Novato, CA). The average series resistance (R_s) in whole-cell configuration was 5.71 ± 0.28 M for TG4 cells (n = 34) and 5.99 ± 0.39 M for WT cells (n = 25), and routinely compensated ~70% in our experiments. To selectively examine I_Ca, cells were voltage-clamped at 40 mV to inactivate the sodium and T-type Ca²⁺ channels. Potassium currents were inhibited by appropriate blockers in the extracellular HEPES buffer solution (4 mM 4-aminopyridine, 5.4 mM CsCl substituted for KCl in standard HEPES buffer solution) and in the pipette solution containing: 100 mM CsCl, 10 mM NaCl, 20 mM tetraethylammonium chloride 20, 10 mM HEPES, 5 mM MgATP, and 5 mM EGTA; pH was adjusted to 7.2 with CsOH. In some experiments to simultaneously record I_Ca and cell contraction, EGTA was omitted from the pipette solution and normal HEPES buffer constituted the extracellular solution. I_Ca was elicited by 300-ms pulses from a holding potential of 40 mV to test potentials from 30 to +50 mV in 10-mV increments at 0.1 Hz at 23°C. To monitor drug effects, I_Ca elicited by a depolarization from 40 to 0 mV was continuously recorded. The amplitude of I_Ca was measured as the difference between the peak inward current and that at the end of 300-ms pulse. The decay of I_Ca was fitted to a biexponential function:
Where _f and _s are the fast and slow inactivation time constants; A₀ is a constant; and A_f and A_s are amplitudes of fast and slow current components, respectively.

Measurement of cAMP Accumulation. Cardiac membranes were prepared as previously described (Xiao et al., 1998). cAMP levels were assayed by the radioimmunoassay. Briefly, 10 µl of membrane vesicles (20 µg total protein) was added to a 40-µl reaction mixture to make a final concentration of 4 mM Tris-EDTA and 10 µM Ro 20-1724 (an inhibitor of phosphodiesterase IV) with or without 0.5 µM ICI 118,551 (ICI is a ₂AR inverse agonist). The reaction was performed for 15 min at 37°C and 25 µl of supernatant was assayed using a cAMP ³H assay kit obtained from Amersham (Arlington Heights, IL). Protein was measured using the Bradford method (Bio-Rad, Richmond, CA) with BSA as the standard.

Materials. PTX, tetrodotoxin, forskolin, isoproterenol hydrochloride, norepinephrine (NE), prazosin, and Ro 20-1724 were purchased from Sigma Chemical Co. (St. Louis, MO). Rp diastereomers of 8-(4-chlorophenylthio)-cAMP (Rp-CPT-cAMPS) was purchased from Biolog Life Science Institute (La Jolla, CA). cAMP assay kits were purchased from Amersham. Zinterol was kindly supplied by Bristol-Myers (Evansville, IN); ICI was kindly supplied by Imperial Chemical Industry (London, United Kingdom). CGP20712A (CGP) was kindly supplied by Ciba-Geigy Corp. (Basel, Switzerland).

Data Analysis. Data are reported as mean ± S.E.M. Student's t test was used to test for differences between TG4 and WT groups and for PTX-treated and nontreated groups; a paired t test was used for assessing the significance of drug effects. A value of P < .05 was considered to be statistically significant.

	Results

Top Summary Introduction Experimental Procedures Results Discussion References

In the absence of exogenous ₂AR agonists, the basal cAMP level was increased by 2.5-fold in TG4 relative to WT hearts (Fig. 1A). Concomitantly, basal contraction amplitude was enhanced by 1.9-fold in single ventricular myocytes isolated from TG4 mice (Fig. 1B). A ₂AR inverse agonist, ICI (5 × 10⁷ M), which had no significant effect on either basal cAMP or contractility in WT mice, reduced the baseline cAMP (Fig. 1A) and contractility of TG4 cells (Fig. 1B) to levels similar to those of WT littermates. These data are in agreement with previous observations that ICI depresses the elevated basal adenylyl cyclase activity, heart rate and cardiac contractility in vivo and in isolated atria (Milano et al., 1994; Bond et al., 1995; Du et al., 1996). Therefore, the results so far support the notion of spontaneous ₂AR activation in the absence of an agonist (Chidiac et al., 1994; Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999) and indicate that ₂-R* augments cAMP production and cardiac contractility, as is the case for ligand-induced ₂AR stimulation (Xiao and Lakatta, 1993; Xiao et al., 1994, 1995; Altschuld et al., 1995; Zhou et al., 1997). If ₂-R* and ₂-LR* were functionally equivalent, as predicted by the two-state model, the L-type Ca²⁺ channel, a key target effector of ₂-LR* signaling, would be modulated by ₂-R* in a similar fashion, i.e., baseline I_Ca in TG4 cells would be expected to be tonically elevated and sensitive to ICI. To our surprise, basal I_Ca was not elevated in TG4 cells (see below). Furthermore, although ICI (5 × 10⁷ M) rapidly and reversibly attenuated the augmented baseline contraction amplitude in TG4 ventricular myocytes (Fig. 2A), it had virtually no effect on the amplitude (Fig. 2B; 97.2 ± 3.4% of control, n = 9) and time course (Fig. 2C) of I_Ca in TG4 cells. This result was further confirmed by the simultaneous recording of I_Ca and contraction using the EGTA-free pipette solution. As shown in Fig. 3, ICI induced a marked decrease in cell contraction amplitude without any change of I_Ca in the same TG4 cell.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Comparison of the basal cAMP (A) and contractility (B) in the 2AR TG4 mice and in WT mice. Both basal cAMP and contraction amplitude are significantly increased in TG4 as compared with that of WT mice, and both increases can be reversed by a 2AR inverse agonist, ICI 118,551 (ICI, 5 × 107 M); n = 3 for cAMP measurements; n = 12 and 9 for contraction measurements in WT and TG4 cells, respectively. *P < .01 for TG4 without ICI group compared with other groups.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   A 2AR inverse agonist, ICI (5 × 107 M), depresses the basal contraction but not ICa in TG4 cardiomyocytes. A, an example of the effect of ICI on basal contraction amplitude. Top, a continuous chart recording of cell length. An upward deflection indicates cell shortening. Bottom, the twitch is displayed at higher resolution at times indicated in top panel. A downward deflection indicates cell shortening. B, typical continuous recording of ICa in response to ICI. ICa is elicited every 30 s by 300-ms pulses from 40 to 0 mV. C, superimposed traces of ICa recorded before and after exposure to ICI at times indicated in B.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   A typical example of simultaneous recording of TG4 cell contraction and ICa in response to the 2AR inverse agonist, ICI (5 × 107 M) or a PKA inhibitor, Rp-CPT-cAMPS (104 M) under the whole-cell voltage clamp condition without EGTA in the pipette. The voltage clamp protocol is shown as the inset. Shortening of cell length is shown in the upper panel and ICa in the lower panel. Similar results were obtained in three other cells.

The differential effects of ₂-R* on I_Ca and contractility are in sharp contrast to the traditional views that the L-type Ca²⁺ channel is an obligatory effector of ₂AR signaling (Xiao and Lakatta, 1993; Cerbai et al., 1995; Altschuld et al., 1995; Zhou et al., 1997). The results also raise doubts as to whether the ₂-R* effect to augment contractility in TG4 myocytes even requires the classical cyclase-cAMP-PKA signaling. To directly address this issue, we used an inhibitory cAMP analog, Rp-CPT-cAMPS, to specifically block PKA activation. As shown in Fig. 3, similar to the effect of the inverse agonist ICI, Rp-CPT-cAMPS reversed the ₂-R* effect on contraction without affecting the simultaneously recorded I_Ca. This observation indicates that the ₂-R*-stimulated inotropic effect in TG4 cells depends largely on ₂-R*-elicited cAMP signaling, as does ₂-LR* (Zhou et al., 1997; Skeberdis et al., 1997; Xiao et al., 1999). Thus, the inability of ₂-R* to modulate L-type Ca²⁺ channels may be attributed to either a qualitative difference between ₂-R* and ₂-LR*, or to an alteration in L-type Ca²⁺ channels of TG4 cells (see below).

To further characterize the L-type Ca²⁺ channel properties in TG4 cells, whole-cell I_Ca amplitude, current-voltage relation, and inactivation kinetics were systematically examined in both TG4 and WT ventricular myocytes. Figure 4A shows typical traces of I_Ca elicited by a depolarization from 40 to 0 mV in a WT and a TG4 myocyte in the absence of any ₂AR ligands. The baseline I_Ca in TG4 and WT cells are virtually indistinguishable in amplitude and time course (Fig. 4A), consistent with the absence of ICI-sensitive (₂-R*) component of I_Ca described above. The average amplitude of I_Ca at 0 mV was 1.01 ± 0.05 nA in TG4 (n = 34) and 1.03 ± 0.07 nA in WT cells (n = 38). Rundown of I_Ca was not significantly different between these two groups (12.4 ± 4.9 and 14.1 ± 6.2% at 10 min for TG4 and WT cells, respectively; n = 3 for both groups). Because there was no significant difference in cell membrane capacitance (166 ± 10 pF, n = 34, in TG4 cells versus 161 ± 12 pF, n = 38, in WT cells), the density of I_Ca (i.e., I_Ca normalized by capacitance) was also similar in TG4 and WT groups (6.73 ± 0.43 pA/pF, n = 34 and 6.86 ± 0.49 pA/pF, n = 38, respectively). The similarity in membrane capacitance between TG4 and WT cells is consistent with a previous report that no cellular hypertrophy occurs in 2- to 4-month-old TG4 hearts (Milano et al., 1994; Xiao et al., 1999).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Properties of basal L-type Ca2+ current (ICa) recorded in single ventricular myocytes isolated from TG4 and WT mice. A, representative traces of ICa recorded from TG4 and WT cells. Inset, voltage clamp protocol to elicit ICa. B, current density-voltage curves obtained from TG4 and WT cells. C, relationship between voltage and inactivation time constants of ICa in TG4 and WT cells. The decay of ICa is fitted to the sum of two exponentials (see Experimental Procedures); n = 19 to 20 for data presented in B and C.

Next, we determined the current-voltage relation of I_Ca in both TG4 and WT myocytes. Cells were depolarized from a holding potential of 40 mV to various test potentials from 30 to +50 mV in 10-mV increments. Over the entire voltage range examined, the I_Ca density-voltage relations in TG4 and WT cells overlapped (Fig. 4B), indicating that voltage-dependent activation of L-type Ca²⁺ channel in TG4 cells was unchanged as compared with WT controls. Furthermore, I_Ca inactivation time constants (_f and _s) and the voltage-dependence of _f or _s of WT cells were similar to those of TG4 cells (Fig. 4C); likewise, there is no difference in the amplitude proportion of the two exponential components between these two groups (A_f/A_s = 1.24 ± 0.08 at 0 mV, n = 20, in TG4 versus 1.19 ± 0.16, n = 19, in WT). Therefore, no measured parameters of I_Ca, including amplitude, voltage-dependence, and inactivation kinetics were altered by spontaneous ₂AR activation in TG4 cardiac myocytes.

If L-type Ca²⁺ channels in TG4 cells were somehow modified via compensatory mechanisms so that I_Ca could no longer respond to ₂-R*-mediated cAMP signaling, the I_Ca response to any other cAMP signaling should be similarly blunted. However, forskolin, an activator of adenylyl cyclase, induced a robust increase in the Cd²⁺-sensitive I_Ca in TG4 cells (Fig. 5, A and B). More importantly, the dose-response curves of I_Ca to forskolin in TG4 and WT cells virtually overlapped, with no significant difference in EC₅₀ (3.97 × 10⁷ M for WT and 5.96 × 10⁷ M for TG4; P > .05, Fig. 5C). Thus, the sensitivity of cardiac L-type Ca²⁺ channel to cAMP-PKA modulation remains intact in TG4 mice.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Response of ICa to a 2AR agonist, zinterol, or an adenylyl cyclase activator, forskolin, in PTX-untreated cardiomyocyte from TG4 mice. A, time course of changes in the peak amplitude of ICa. ICa is activated by 300-ms depolarization pulses from a holding potential of 40 to 0 mV at 0.1 Hz. Note that ICa is not affected by zinterol at 106 M, but is markedly increased by forskolin at 106 M, and that ICa is abolished by 5 × 105 M Cd2+. B, selected current traces recorded before or after exposure to different drugs. Letters "a" to "e" correspond to those time points marked in A. C, dose-response curves of ICa to forskolin in myocytes from TG4 or WT hearts. The values of EC50 in WT (3.97 × 107 M) and TG4 (5.96 × 107 M) are not significantly different (P > .05). Each point represents mean ± S.E.M. of results from four to eight cells. Data are expressed as percentage of control value (C). Control values of ICa are 0.95 ± 0.09 nA for TG4 (n = 17) and 0.91 ± 0.07 nA for WT (n = 20).

Our recent studies have shown that cardiac ₂AR couples to the PTX-sensitive inhibition proteins, (G_i) G_i2 and G_i3 (Xiao et al., 1995, 1999), and that this coupling partially offsets the ₂AR agonist-mediated contractile response in rat myocytes (Xiao et al., 1995) and completely negates the ₂AR agonist-mediated contractile (Xiao et al., 1999) and I_Ca responses (Fig. 5, A and B) in TG4 and WT murine ventricular myocytes. Therefore, it is reasonable to assume that an excessive G_i coupling to ₂-R* could be involved in the inability of ₂-R* to modulate I_Ca. To test this hypothesis, baseline I_Ca was re-examined in PTX-treated cells and compared with that in PTX-untreated cells. Figure 6B shows that in TG4 cells, PTX treatment had no significant effect on the baseline I_Ca amplitude or its current-voltage relation. Similar results were also obtained in WT cells (Fig. 6A). Moreover, even in PTX-treated TG4 cells, neither the amplitude nor the kinetics of the basal I_Ca were affected by ICI (data not shown). These results suggest that G_i proteins are not involved in the unresponsiveness of I_Ca to ₂-R*.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of PTX treatment on the baseline ICa and the response of ICa to zinterol in ventricular myocytes from WT and TG4 mice. A and B, current-voltage curves obtained in PTX-untreated (open symbols, dashed line) and PTX-treated cells (filled symbols, solid line) from WT (A) and TG4 (B) cells. C, current-voltage curves obtained before (open symbols, dashed lines) and 5 min after exposure to zinterol (106 M, filled symbols, solid lines) in PTX-treated TG4 cells. D, time course of changes in peak magnitude of ICa after exposure to zinterol (106 M) and the blockade of zinterol's effect by ICI (5 × 107 M) in a representative PTX-treated TG4 cell. In A-C, each symbol represents the means ± S.E.M. from 5 to 20 cells. *P < .05 compared with values before zinterol in PTX-treated TG4 cells.

Although G_i inhibition failed to rescue I_Ca response to ₂-R*, in the same TG4 cells, PTX permitted ₂-LR* induced by zinterol to significantly enhance I_Ca (Fig. 6, C and D). The PTX rescued I_Ca response to ₂-LR* in TG4 cells (149 ± 12% of control, at 0 mV, n = 8) was comparable with that of WT cells (153 ± 11% of control, at 0 mV, n = 4). In addition, the I_Ca-voltage relation was shifted leftward by zinterol (V_1/2 was 16.58 ± 1.33 and 23.03 ± 1.67 mV in the absence and presence of zinterol, respectively, P = .01, Fig. 6C), in agreement with previous observations in rat ventricular myocytes (Xiao and Lakatta, 1993). However, neither the inactivation kinetics (_f, 101 ± 8% of control, _s, 108 ± 3% of control), nor the ratio of A_f/A_s (95 ± 19% of control, n = 5) were significantly altered by zinterol in PTX-treated TG4 cells. Figure 6D shows that the I_Ca response to zinterol in a PTX-treated TG4 cell was completely blocked by the ₂AR-selective antagonist, ICI at 5 × 10⁷ M (96.2 ± 6.2% of control, n = 5, P > .05 versus control). Thus, PTX treatment permits ₂-LR*, but not ₂-R*, to modulate L-type Ca²⁺ channel activity in TG4 heart.

Although in mouse cardiac myocytes ₁-AR is unable to couple to G_i proteins, as manifested by the G protein photoaffinity labeling profile (Xiao et al., 1999), previous studies in guinea pig (Hool and Harvey, 1997) raised doubt as to whether the PTX rescued effect of zinterol is related to the activation of ₁AR. We therefore examined the effect of ₁AR stimulation in the presence and absence of PTX treatment in TG4 myocytes. Interestingly, ₁AR agonist NE even at maximal concentration (NE 10⁷ M) plus prazosin 10⁶ M (Korzick et al., 1997) did not induce a discernible increase in I_Ca of TG4 cells, whereas it markedly increased I_Ca in WT myocytes (Fig. 7, A and B). The absence of I_Ca response to ₁AR stimulation is consistent with previous observations on the loss of contractile response to ₁AR stimulation by either NE plus prazosin or isoproterenol plus the ₂AR blocker, ICI (Bond et al., 1995; Du et al., 1996). Whereas PTX treatment fully rescued the contractile (Xiao et al., 1999, also see Fig. 7C) and I_Ca (Fig. 6) response to ₂AR agonist stimulation, it was unable to restore contractile and I_Ca response to ₁AR stimulation (Fig. 7). In addition, in TG4 cells, the PTX-restored contractile response to a mixed AR agonist, isoproterenol 10⁶ M, was specifically inhibited by a ₂AR antagonist, ICI 10⁷ M, but not by a ₁AR antagonist, CGP 3 × 10⁷ M (Fig. 7C). This further corroborates our previous notions that, unlike ₂AR, ₁AR does not couple to G_i protein(s) in mouse myocardium (Xiao et al., 1999).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   ICa and contractile response to AR stimulation in mouse ventricular myocyte. A, typical current traces recorded before or after 1AR stimulation by norepinephrine (NE, 107 M) plus prazosin (Praz, 106 M) in representative WT, TG4, and PTX-treated TG4 myocytes. B, average increase in ICa elicited by a depolarization pulse from 40 to 0 mV in response to 1AR stimulation. Control values of ICa are 1.03 ± 0.14 nA for WT (n = 7), 1.09 ± 0.06 nA for TG4 (n = 5), and 1.21 ± 0.15 nA for PTX-treated TG4 myocytes (n = 3). *P < .01 versus control. C, a typical example of continuous chart recording of cell length following AR stimulation by isoproterenol (ISO, 106 M) in a PTX-treated TG4 cell. An upward deflection indicates cell shortening. The 2AR antagonist, ICI118511 (ICI, 107 M), but not the 1AR antagonist, CGP20712A (CGP, 3 × 107 M), specifically inhibited the PTX-rescued contractile response to ISO. Similar results are observed in other 10 cells.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

₂-R* Does Not Regulate I_Ca. The presence of ₂-R* in the TG4 heart is evidenced by the elevated basal adenylyl cyclase activity (Milano et al., 1994) and cAMP production (Fig. 1A), the enhanced cardiac contractility (Milano et al., 1994; Bond et al., 1995; Du et al., 1996; Rockman et al., 1996; Xiao et al., 1999) (Fig. 1B), and the blockade of these augmentations by the inverse ₂AR agonist, ICI (Milano et al., 1994; Bond et al., 1995; Du et al., 1996; Xiao et al., 1999) (Figs. 2 and 3). In the present study, we have provided direct evidence that ₂-R*-mediated modulation of cardiac contractility is largely cAMP-PKA-dependent, because it is sensitive to the PKA inhibitor Rp-CPT-cAMPS (Fig. 3). The most surprising and unexpected finding of this study is that baseline I_Ca in TG4 cardiac myocytes is not increased or altered by ₂-R* (Fig. 4). The simplest explanation for this observation would be that ₂-R*-directed signaling is totally diverted from the L-type Ca²⁺ channels. However, the interpretation for the results obtained from the transgenic model may not be so straightforward, because compensatory changes have been documented in TG4 hearts, e.g., down-regulation of the SR protein phospholamban (PLB) (Rockman et al., 1996) and up-regulation of G_i proteins (R-P.X., unpublished data). Several additional experiments have therefore been undertaken to explore alternative possibilities.

Differences between ₂-R*- and ₂-LR*-Mediated Signaling. In contrast to the prediction of the two-state receptor model, the differential regulation of I_Ca by ₂-R* and ₂-LR* suggests that the liganded and unliganded active ₂ARs are different active receptor species, likely having different conformations and initiating distinct postreceptor signaling pathways. Several lines of additional evidence support this hypothesis. First of all, whereas ₂-R* in TG4 heart significantly increases the baseline contractility, ₂-LR* induced by zinterol or isoproterenol at maximal concentrations are unable to further increase contraction amplitude (Milano et al., 1994; Du et al., 1996; Xiao et al., 1999), even though the basal contractility is not at the maximum contractile state yet (Du et al., 1996; Xiao et al., 1999). Secondly, ₂-R*, unlike ₂-LR*, does not couple to G_i proteins, as reflected by the lack of a PTX effect on the basal I_Ca (Fig. 6) and by immunoprecipitation data on receptor-G protein interaction (Gurdal et al., 1997). Finally, it has recently been shown that in rat and mouse cardiac myocytes, multiple active conformational states of ₂AR can be induced by different ₂AR ligands (R-P.X., unpublished data). Similar observations have been reported previously for ₂AR and other G protein-coupled receptors in transfected cells (e.g., Eason et al., 1994) or artificial lipid vesicles (Gether et al., 1997). The present finding that ₂-R* differs from ₂-LR* is in general agreement with the emerging concept of multiple active receptor states for a given receptor.

Possible Mechanism for ₂-R* to Augment Cardiac Contractility. Cardiac contractility is an integrated parameter determined by several effectors involved in the E-C coupling cascade. Although I_Ca is unaffected by ₂-R*, the increase in the adenylyl cyclase activity and cAMP production may modulate the E-C coupling cascade by PKA-dependent phosphorylation of target proteins downstream of L-type Ca²⁺ channels, e.g., the SR Ca²⁺ release channels, SR membrane protein PLB, and some contractile proteins. Indeed, our preliminary observations have shown that in TG4 ventricular myocytes, the frequency of "Ca²⁺ sparks" (i.e., the elementary SR Ca²⁺ release events) and the amplitude of whole cell Ca²⁺ transients are markedly increased in TG4 cells, and that both are sensitive to ICI. In addition, there is an adaptive down-regulation of PLB expression in TG4 hearts (Rockman et al., 1996) and thereby less basal inhibition of the SR Ca²⁺ pump in cardiac cells from these transgenic animals. Thus, the enhanced SR Ca²⁺ recycling may be sufficient to account for the augmentation of baseline contractility in TG4 heart. Regardless of the specific mechanism, the suppression of the enhanced basal contractility by Rp-CPT-cAMPS (Fig. 3) indicates that the ₂-R*-elicited contractile effect is largely cAMP/PKA dependent.

Loss of ₁AR Function Associated with ₂AR Overexpression. Although both ₁AR and ₂AR coexist in mouse ventricular myocyte, the function of ₁AR is undetectable in ₂AR overexpression transgenic (TG4) murine heart, as shown by the absence of I_Ca (Fig. 7, A and B) or contractile response (Fig. 7C; also see Bond et al., 1995; Du et al., 1996) to ₁AR stimulation by either NE plus prazosin or isoproterenol plus the ₂AR blocker, ICI. In contrast, in WT mouse ventricular myocyte, ₁AR stimulation produced a dose-dependent increase in contraction amplitude (Korzick et al., 1997) and I_Ca (Fig. 7, A and B). In TG4 myocytes, PTX treatment only rescues the contractile and I_Ca responses to ₂AR agonists, but not to ₁AR agonists (Fig. 7; also see Xiao et al., 1999). Although the exact mechanism for the loss of ₁AR function in TG4 heart is unknown, this phenotype seems to be linked to the overexpression of ₂AR, because the ₁AR function also disappeared in rat C₆ glioma cells overexpressed ₂AR (Zhong et al., 1996). These results indicate a complex interaction between AR subtypes (Zhong et al., 1996).

₂-AR Stimulation in TG4 Hearts at Different Ages. Recent studies have shown that I_Ca density is increased in embryonic/neonatal TG4 myocytes (An et al., 1999), but decreased in 3- to 8-month old TG4 mouse heart cells (Heubach et al., 1999) as compared with age-matched controls. In the present study, we found no evidence for any difference in I_Ca characteristics between transgenic and WT cells from young adult animals (2-3 months old). This apparent discrepancy may reflect an age-related change in AR signaling cascade. In nontransgenic rat, there are striking developmental changes with respect to ₂AR agonist sensitivity and functions (Kuznetsov et al., 1995, Xiao et al., 1998), perhaps due to a developmental changes in ₂AR-G_i coupling. In this scenario, it is not surprising that spontaneous ₂AR activation may exhibit differential functions at different stages of development. Alternatively, it is possible that some compensatory changes (e.g., expression of L-type Ca²⁺ channel) may occur progressively as a result of the receptor overexpression, rendering divergent and even conflicting phenotypes at different ages. Nevertheless, as discussed above, a compensatory change in Ca²⁺ channel sensitivity to cAMP-PKA signaling cannot account for the inability of ₂-R*s to regulate I_Ca in the young mouse heart.